Sprayable barrier and methods for prevention of postoperative adhesions

ABSTRACT

Biologically acceptable surgical barrier materials are comprised of a polyelectrolytic complex of chitosan and sodium alginate. The chitosan is deacetylated in an amount between about 40 to about 60% and has a molecular weight (Mw) between 50,000 and 375,000 g/mol. The barrier materials may be formed by mixing a two-component material system whereby one component comprises the chitosan and a second component comprises the sodium alginate and directing such a mixture (e.g., via air-assisted spray nozzle) toward a surgical site in need of the material. A polyelectrolytic complex of the chitosan and sodium alginate will thereby form in situ. Suitable ionic cross-linkers may be provided in the individual components, e.g., calcium chloride with the chitosan component and sodium tripolyphosphate with the sodium alginate component.

GOVERNMENT RIGHTS

This invention was made with Government support under NIH Phase IProgram 2 R44 GM074373-01, NIH Phase II Program 2 R44 GM074373-02, NIHPhase I Program 1 R43 GM105142-01 and NIH Phase II Program 5 R44GM105142-03. The Government has certain rights to the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is based on and claims priority benefits of U.S.Provisional Application Ser. No. 62/482,294 filed on Apr. 6, 2017, theentire contents of which are expressly incorporated hereinto byreference.

FIELD

The disclosed embodiments herein relate generally to sprayable materialsthat prevent adhesions form forming postoperatively at a surgical site.

BACKGROUND AND SUMMARY

Injury to tissue during surgery, trauma, infection, or other forms ofinflammation will lead to the initiation of wound healing. If the fibrinmatrix deposited during the initial stages of wound healing formspermanent contacts with neighboring tissue, it can form a fibrous strandthat will capture fibroblasts and mesothelial cells until the fibrousstrand develops into a firm, vascularized adhesion. Adhesion formationafter surgery (e.g., intra-abdominal surgery) poses a significant healthrisk and has been cited as a leading cause of small bowel obstructionand female infertility. Methods to prevent these adhesions from formingare currently limited to inefficient liquid treatments that are quicklyresorbed, or solid barrier films, which are difficult to place and areoften restricted to use at the incision site, ultimately leaving thebulk of the abdominal contents unprotected.

It would therefore be highly desirable if a barrier material could beprovided that would effectively prevent such tissue adhesionspostoperatively at a surgical site. It is towards fulfilling such a needthat the embodiments disclosed herein are directed.

In general, the embodiments disclosed herein are directed toward asprayable hydrogel barrier that adheres to the internal tissues, e.g. ofthe patient's abdominal cavity, providing protection from adhesions tosusceptible internal surfaces. The barrier gel disclosed herein isprincipally based on the interaction of two naturally-occurringbiopolymers that together form a polyelectrolyte complex and is coupledwith ionic crosslinkers to enhance the integrity of the resultant gel.The disclosed barrier gels are both biocompatible and biodegradable, andprovide more complete protection than is available from currentlymarketed adhesion-prevention products. The barrier gel is applied bysimultaneously spraying the two components over the injured ordesiccated tissue. Upon interaction, a hydrogel begins to form that willionically crosslink within less than 1 minute. The sprayable componentsare easily visualized during application due to the color in one of thecomponents and may therefore be used satisfactorily for laparoscopicapplications and open-abdominal procedures.

These and other aspects of the present invention will become more clearafter careful consideration is given to the following detaileddescription of a presently preferred exemplary embodiment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference will be made to the accompanying drawing Figures, wherein:

FIG. 1 is a graph showing mass loss data of hydrogel formulation samplessubmerged in PBS or lysozyme solutions which suggests that there shouldbe complete degradation of the sample within a 28 day window;

FIGS. 2A and 2B are bar graphs showing the adhesion score (FIG. 2A) andscaled adhesion score (FIG. 2B) demonstrating that the local application(hydrogel material contained over two injury sites) of sprayablehydrogel material using an air-assisted spray tip results instatistically significant decrease in the incidence and strength ofprimary adhesions; and

FIGS. 3A and 3B are bar graphs showing the adhesion score (FIG. 3A) andscaled adhesion score (FIG. 3B) demonstrating that the full application(hydrogel material applied over inured sites and neighboring tissue) ofsprayable hydrogel material results in a slight improvement as comparedto local application.

DETAILED DESCRIPTION

The sprayable barrier materials disclosed herein will necessarilyinclude a chitosan component and a sodium alginate component. Eachcomponent may be provided separately of the other and then mixedtogether using a suitable atomizing spray system immediately prior toapplication at the desired tissue site. If mixture of the components isdone by means of an atomizing spray system, then atmospheric air or aninert gas, e.g., carbon dioxide (CO₂), may be employed as an atomizingmedium at an atomizing pressure of between about 5 psi to about 15 psi,typically 10 psi(+/−3 psi).

Each component of the sprayable barrier materials will be furtherdescribed hereinbelow.

A. Chitosan Component

Chitosan is a linear polysaccharide derived from chitin, a biopolymerfound in the exoskeleton of shrimp and crabs and also produced fromcertain fungal sources. Chitosan is generated from chitin by theconversion of N-acetyl groups to amino groups and is soluble in diluteacid solutions. The protonated amine group lends the chitosan apolycationic nature that may promote mucoadhesion to tissue and allowsfor controlled interaction with negatively charged ions.

The chitosan that may be employed in the practice of the barriermaterials disclosed herein can be obtained commercially from a number ofsources in powdered form. The commercially obtained chitosan issubjected to the following controlled processing in order to obtainchitosan having 40-60% deacetylation thereby providing the requiredwater solubility.

For such processing, the chitosan is suspended in a 50% sodium hydroxidesolution that has been purged with nitrogen and heated to 120° C. for2.5 hours. The chitosan is then filtered and rinsed until neutral,resulting in 95% deacetylated chitosan. After this deacetylationtreatment, the chitosan can be easily dissolved into dilute acetic acid(0.1 Molar) at 2% w/v. Acetic anhydride is then added dropwise at 0.75%v/v and stirred for one hour to induce reacetylation of the chitosan to40-60% deacetylation. Upon precipitation with the addition of 3-4×volumes of acetone, the chitosan is centrifuged to remove it fromsolution, and the pelleted treated chitosan product is lyophilized toproduce a water-soluble powder.

Deacetylation and reacetylation processing may be accomplished usinghigh molecular weight (M_(w)=310-375 kDa), medium molecular weight(M_(w)=190-310 kDa), and low molecular weight (M_(w)=50-190 kDa)chitosan. First-derivative UV-Vis characterization may be used toquantify the degree of acetylation throughout the chitosan treatmentprocess. It has been determined that despite the different startingdegrees of acetylation, the first deacetylation treatment bringssubstantially all chitosan samples to a level of 95% deacetylation,after which the 0.75% acetic anhydride reacetylation treatmentconsistently yields chitosan samples having a deacetylation level of40-60%. Low M_(w) chitosan may be difficult to precipitate and collectafter reacetylation treatment. Medium M_(w) and high M_(w) chitosan aretypically easier to collect. Preferably medium M_(w) chitosan issubjected to the deacetylation-reacetylation treatment as it has beenshown to form the hydrogels with the most desirable properties whendissolved at 0.5-1.5% w/v in deionized water. The chitosan productshould have a molecular weight (M_(w)) between 50,000 and 375,000 g/mol.

The chitosan component and/or the sodium alginate component may beprovided with suitable additives, for example, antifungal agents,coloring agents, cross-linking agents, therapeutic agents and the like.By way of example, sodium benzoate is preferably included within the twocomponents to prevent mold growth in the solutions.

Calcium chloride may also be included in the chitosan component solutionto serve as an ionic crosslinker to improve the gelation of the mixedbarrier material. Calcium ions do not interact with chitosan insolution, but upon mixing with the sodium alginate component, thecalcium will then act as a relatively strong ionic crosslinker of thealginate. This ionic cross-linking results in a gel with the physicalproperties necessary for keeping injured tissues physically separatedfrom one another during healing. The calcium chloride may be present inthe chitosan component solution in an amount less than 2 wt. %, based ontotal solution weight, e.g., 0 to 2 wt. %, with between about 1 to about1.5 wt. % calcium chloride content being especially preferred forachieving the best crosslinking for gel physical properties withoutimpeding degradation over a reasonable time frame (e.g., 14-28 days).

B. Alginate Component

Sodium alginate is the sodium salt of alginic acid, commonly referred toas alginate, an anionic polysaccharide found in the cell walls of algae.Alginate is a linear copolymer that is water soluble and associates withcalcium to form an ionically crosslinked polymer network. Alginate isused throughout the food and drug industry as a thickening agent. Thealginate should have a molecular weight of from about 75,000 to about500,000 g/mol.

Alginate and chitosan associate with one another to form apolyelectrolyte complex in which the positive charges on the aminogroups in chitosan associate with the negatively charged carboxylategroups on alginate to form a polymeric association and gelationreaction. The inclusion of an ionic crosslinker in the chitosancomponent solution (e.g., calcium chloride to interact with alginate)and sodium tripolyphosphate in the alginate component solution (e.g., tointeract with chitosan) serves to further strengthen the integrity ofthe resultant gel. Sodium alginate is easily dissolved in water from0-2% w/v with gentle heating and agitation. Used as a thickening agent,it has been found in these solutions to be most workable for the desiredsprayable application when the sodium alginate component is present inthe solution in an amount between about 0.25 to about 0.5% w/v.

Sodium tripolyphosphate (NaTPP) is the sodium salt of the polyphosphatepenta-anion. With multiple negative charges present, NaTPP acts as astrong ionic crosslinker for chitosan, but does not interact with thealginate in solution prior to spraying. It has been determined thatNaTPP concentrations between about 0.5 to about 1.5 wt. %, based on thetotal weight of the alginate component solution, form gels of desiredintegrity.

Methylene blue is a dark green powder that yields a blue solution whendissolved in water. Methylene blue is a component of a frequentlyprescribed urinary analgesic/anti-infective medicine and has beensuggested to help in the prevention of adhesion formation after surgery.Methylene blue is used in the sprayable barrier materials as a visualaid, allowing the surgeon to visually determine where the gel has beensprayed and which tissues remain unprotected. The amount of methyleneblue that may be employed as a visual marker in the alginate componentsolution is typically less than about 0.01 wt. %, based on the totalsolution weight. At such levels, a sufficient blue color needed forvisualization of the sprayed gel is provided. Other biologicallyacceptable colorants, e.g., dimethyl-methylene blue which provides alighter purple color, may also satisfactorily be employed.

EXAMPLES

The following formulations shown in Table 1 below may be employed toobtain sprayable gel barrier materials in accordance with the invention:

TABLE 1 Sprayable hydrogel formulations (all percentages are weightpercent based on the total weight of the final formulation or componentsolution as appropriate.) Syringe 1 Syringe 2 Final Calcium SodiumMethlyene Formulation Formulation Volume Chitosan Chloride SolventVolume Alginate Blue NaTPP Solvent  1 0.5% 3 mL 1% Deionized 3 mL 1%0.1% Deionized (1-959-61) Chitosan (MMW Water Water 0.5% 1xDA-RA,Alginate DD = 25) 0.05% Methylene Blue  2 0.5% 3 mL 1% Deionized 3 mL0.5% 0.1% Deionized (2-959-62) Chitosan (MMW Water Water 0.25% 1xDA-RA,Alginate DD = 25) 0.05% Methylene Blue  3 0.5% 3 mL 1% 10% Deionized 3mL 0.5% 0.1% 10% Deionized (3-959-63) Chitosan (MMW Water Water 0.25%1xDA-RA, Alginate DD = 25) 0.05% Methylene Blue 5% Calcium Chloride 5%NaTPP  4 0.5% 3 mL 1% 1% Deionized 3 mL 0.5% 0.1% 1% Deionized(4-959-64) Chitosan (MMW Water Water 0.25% 1xDA-RA, Alginate DD = 25)0.05% Methylene Blue 0.5% Calcium Chloride 0.5% NaTPP  5 0.5% 3 mL 1%1.5% Deionized 3 mL 0.5% 0.1% 1.5% Deionized (5-959-86) Chitosan (HMWWater Water 0.25% 1xDA-RA, Alginate DD = 46) 0.05% Methylene Blue 0.75%Calcium Chloride 0.75% NaTPP  6 0.5% 3 mL 1% 1.5% Deionized 3 mL 0.25%0.1% 1.5% Deionized (6-959-92) Chitosan (HMW Water Water 0.125% 1xDA-RA,Alginate DD = 46) 0.05% Methylene Blue 0.75% Calcium Chloride 0.75%NaTPP  7 0.5% 3 mL 1% 1.5% Deionized 3 mL 0.5% 0.05% 1.5% Deionized(7-959-93) Chitosan (HMW Water Water 0.25% 1xDA-RA, Alginate DD = 46)0.025% Methylene Blue 0.75% Calcium Chloride 0.75% NaTPP  8 0.5% 3 mL 1%1.5% Deionized 3 mL 0.5% 0.05% 1.5% Deionized (8-959-94) Chitosan (HMWWater Water 0.25% 1xDA-RA, Alginate DD = 46) 0.025% Methylene Blue 0.75%Calcium Chloride 0.75% NaTPP  9 0.5% 3 mL 1% Chitosan 1.5% Deionized 3mL 0.5% 0.05% 1.5% Deionized (9-959-95) Chitosan (MMW Water Water 0.25%1xDA-RA, Alginate DD = 47) 0.025% Methylene Blue 0.75% Calcium Chloride0.75% NaTPP 10 0.5% 3 mL 1% 1.5% Deionized 3 mL 0.5% 0.05% 1.5%Deionized (10-959-96) Chitosan (LMW Calcium Water Water 0.25% 1xDA-RA,Chloride Alginate DD = 49) 0.025% Methylene Blue 0.75% Calcium Chloride0.75% NaTPP 11 0.5% 3 mL 1% 1.5% Deionized 3 mL 0.5% 0.01% 1.5%Deionized (11-959-108) Chitosan (MMW Water Water 0.25% 1xDA-RA, AlginateDD = 47) 0.005% Methylene Blue 0.75% Calcium Chloride 0.75% NaTPP 120.75% 1.5 mL 1.5% 1.5% Deionized 1.5 mL 0.5% 0.01% 1.5% Deionized(12-959-124) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate 0.005%Methylene Blue 0.75% Calcium Chloride 0.75% NaTPP 13 0.75% 1.5 mL 1.5%1.5% Deionized 1.5 mL 0.5% 0.025% 2% Deionized (13-959-126) Chitosan(MMW Water Water 0.25% 1xDA-RA) Alginate 0.012% Methylene Blue 0.75%Calcium Chloride 1% NaTPP 14 0.75% 1.5 mL 1.5% 1.5% Deionized 1.5 mL0.5% 0.025% 2% Deionized (14-959-129) Chitosan (HMW Water Water 0.25%1xDA-RA) Alginate 0.012% Methylene Blue 0.75% Calcium Chloride 1% NaTPP15 0.75% 1.5 mL 1.5% 1.5% Deionized 1.5 mL 1% 0.025% 1.5% Deionized(15-959-130) Chitosan (HMW Water Water 0.5% 1xDA-RA) Alginate 0.012%Methylene Blue 0.75% Calcium Chloride 0.75% NaTPP 16 0.75% 1.5 mL 1.5%1.5% Deionized 1.5 mL 1% 0.025% 1.5% Deionized (16-959-135) Chitosan(MMW Water Water 0.5% 1xDA-RA) Alginate 0.012% Methylene Blue 0.75%Calcium Chloride 0.75% NaTPP 17 0.25% 1 mL 0.5% 1.5% Deionized 1 mL0.25% 0.01% 1.5% Deionized (17-995-45a) Chitosan (MMW Water Water 0.125%1xDA-RA) Alginate 0.005% Methylene Blue 0.75% Calcium Chloride 0.75%NaTPP 18 0.25% 1 mL 0.5% 1% e Deionized 1 mL 0.25% 0.01% 1.5% Deionized(18-995-45b) Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005%Methylene Blue 0.5% Calcium Chloride 0.75% NaTPP 19 0.25% 1 mL 0.5% 0.5%Deionized 1 mL 0.25% 0.01% 1.5% Deionized (19-995-45c) Chitosan (MMWWater Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue 0.25% CalciumChloride 0.75% NaTPP 20 0.5% 1 mL 1% n 1.5% Deionized 1 mL 0.25% 0.01%1.5% Deionized (20-995-45d) Chitosan (MMW Water Water 0.125% 1xDA-RA)Alginate 0.005% Methylene Blue 0.75% Calcium Chloride 0.75% NaTPP 210.5% 1 mL 1% n 1% Deionized 1 mL 0.25% 0.01% 1.5% Deionized (21-995-45e)Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue0.5% Calcium Chloride 0.75% NaTPP 22 0.5% 1 mL 1% 0.5% Deionized 1 mL0.25% 0.01% 1.5% Deionized (22-995-45f) Chitosan (MMW Water Water 0.125%1xDA-RA) Alginate 0.005% Methylene Blue 0.25% Calcium Chloride 0.75%NaTPP 23 0.25% 1 mL 0.5% 1.5% Deionized 1 mL 0.25% 0.01% 1% Deionized(23-995-46a) Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005%Methylene Blue 0.75% Calcium Chloride 0.5% NaTPP 24 0.25% 1 mL 0.5% 1%Deionized 1 mL 0.25% 0.01% 1% Deionized (24-995-46b) Chitosan (MMW WaterWater 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue 0.5% CalciumChloride 0.5% NaTPP 25 0.25% 1 mL 0.5% 0.5% Deionized 1 mL 0.25% 0.01%1% Deionized (25-995-46c) Chitosan (MMW Water Water 0.125% 1xDA-RA)Alginate 0.005% Methylene Blue 0.25% Calcium Chloride 0.5% NaTPP 26 0.5%1 mL 1% 1.5% Deionized 1 mL 0.25% 0.01% 1% Deionized (26-995-46d)Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue0.75% Calcium Chloride 0.5% NaTPP 27 0.5% 1 mL 1% 1% Deionized 1 mL0.25% 0.01% 1% Deionized (27-995-46e) Chitosan (MMW Water Water 0.125%1xDA-RA) Alginate 0.005% Methylene Blue 0.5% Calcium Chloride 0.5% NaTPP28 0.5% 1 mL 1% 0.5% Deionized 1 mL 0.25% 0.01% 1% Deionized(28-995-46f) Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005%Methylene Blue 0.25% Calcium Chloride 0.5% NaTPP 29 0.25% 1 mL 0.5% 1.5%Deionized 1 mL 0.25% 0.01% 0.5% Deionized (29-995-46g) Chitosan (MMWWater Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue 0.75% CalciumChloride 0.25% NaTPP 30 0.25% 1 mL 0.5% 1% Deionized 1 mL 0.25% 0.01%0.5% Deionized (30-995-47a) Chitosan (MMW Water Water 0.125% 1xDA-RA)Alginate 0.005% Methylene Blue 0.5% Calcium Chloride 0.25% NaTPP 310.25% 1 mL 0.5% 0.5% Deionized 1 mL 0.25% 0.01% 0.5% Deionized(31-995-47b) Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005%Methylene Blue 0.25% Calcium Chloride 0.25% NaTPP 32 0.5% 1 mL 1% 1.5%Deionized 1 mL 0.25% 0.01% 0.5% Deionized (32-995-47c) Chitosan (MMWWater Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue 0.75% CalciumChloride 0.25% NaTPP 33 0.5% 1 mL 1% 1% Deionized 1 mL 0.25% 0.01% 0.5%Deionized (33-995-47d) Chitosan (MMW Water Water 0.125% 1xDA-RA)Alginate 0.005% Methylene Blue 0.5% Calcium Chloride 0.25% NaTPP 34 0.5%1 mL 1% 0.5% Deionized 1 mL 0.25% 0.01% 0.5% Deionized (34-995-47e)Chitosan (MMW Water Water 0.125% 1xDA-RA) Alginate 0.005% Methylene Blue0.25% Calcium Chloride 0.25% NaTPP 35 0.25% 1 mL 0.5% 1.5% Deionized 1mL 0.5% 0.01% 1.5% Deionized (35-995-47f) Chitosan (MMW Water Water0.25% 1xDA-RA) Alginate 0.005% Methylene Blue 0.75% Calcium Chloride0.75% NaTPP 36 0.25% 1 mL 0.5% 1% e Deionized 1 mL 0.5% 0.01% 1.5%Deionized (36-995-47g) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate0.005% Methylene Blue 0.5% Calcium Chloride 0.75% NaTPP 37 0.25% 1 mL0.5% 0.5% Deionized 1 mL 0.5% 0.01% 1.5% Deionized (37-995-48a) Chitosan(MMW Water Water 0.25% 1xDA-RA) Alginate 0.005% Methylene Blue 0.25%Calcium Chloride 0.75% NaTPP 38 0.5% 1 mL 1% 1.5% Deionized 1 mL 0.5%0.01% 1.5% Deionized (38-995-48b) Chitosan (MMW Water Water 0.25%1xDA-RA) Alginate 0.005% Methylene Blue 0.75% Calcium Chloride 0.75%NaTPP 39 0.5% 1 mL 1% 1% Deionized 1 mL 0.5% 0.01% 1.5% Deionized(39-995-48c) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate 0.005%Methylene Blue 0.5% Calcium Chloride 0.75% NaTPP 40 0.5% 1 mL 1% 0.5%Deionized 1 mL 0.5% 0.01% 1.5% Deionized (40-995-48d) Chitosan (MMWWater Water 0.25% 1xDA-RA) Alginate 0.005% Methylene Blue 0.25% CalciumChloride 0.75% NaTPP 41 0.25% 1 mL 0.5% 1.5% Deionized 1 mL 0.5% 0.01%1% Deionized (41-995-48e) Chitosan (MMW Water Water 0.25% 1xDA-RA)Alginate 0.005% Methylene Blue 0.75% Calcium Chloride 0.5% NaTPP 420.25% 1 mL 0.5% 1% Deionized 1 mL 0.5% 0.01% 1% Deionized (42-995-48f)Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate 0.005% Methylene Blue0.5% Calcium Chloride 0.5% NaTPP 43 0.25% 1 mL 0.5% 0.5% Deionized 1 mL0.5% 0.01% 1% Deionized (43-995-48g) Chitosan (MMW Water Water 0.25%1xDA-RA) Alginate 0.005% Methylene Blue 0.25% Calcium Chloride 0.5%NaTPP 44 0.5% 1 mL 1% 1.5% Deionized 1 mL 0.5% 0.01% 1% Deionized(44-995-49a) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate 0.005%Methylene Blue 0.75% Calcium Chloride 0.5% NaTPP 45 0.5% 1 mL 1% 1%Deionized 1 mL 0.5% 0.01% 1% Deionized (45-995-49b) Chitosan (MMW WaterWater 0.25% 1xDA-RA) Alginate 0.005% Methylene Blue 0.5% CalciumChloride 0.5% NaTPP 46 0.5% 1 mL 1% 0.5% Deionized 1 mL 0.5% 0.01% 1%Deionized (46-995-49c) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate0.005% Methylene Blue 0.25% Calcium Chloride 0.5% NaTPP 47 0.25% 1 mL0.5% 1.5% Deionized 1 mL 0.5% 0.01% 0.5% Deionized (47-995-49d) Chitosan(MMW Water Water 0.25% 1xDA-RA) Alginate 0.005% 0.5% Methylene Blue0.75% Calcium Chloride 0.25% NaTPP 48 0.25% 1 mL 0.5% 1% Deionized 1 mL0.5% 0.01% 0.5% Deionized (48-995-49e) Chitosan (MMW Water Water 0.25%1xDA-RA) Alginate 0.005% Methylene Blue 0.5% Calcium Chloride 0.25%NaTPP 49 0.25% 1 mL 0.5% 0.5% Deionized 1 mL 0.5% 0.01% 0.5% Deionized(49-995-49f) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate 0.005%Methylene Blue 0.25% Calcium Chloride 0.25% NaTPP 50 0.5% 1 mL 1% 1.5%Deionized 1 mL 0.5% 0.01% 0.5% Deionized (50-995-49g) Chitosan (MMWCalcium Water Water 0.25% 1xDA-RA) Chloride Alginate 0.005% MethyleneBlue 0.75% Calcium Chloride 0.25% NaTPP 51 0.5% 1 mL 1% 1% Deionized 1mL 0.5% 0.01% 0.5% Deionized (51-995-50a) Chitosan (MMW Water Water0.25% 1xDA-RA) Alginate 0.005% Methylene Blue 0.5% Calcium Chloride0.25% NaTPP 52 0.5% 1 mL 1% 0.5% Deionized 1 mL 0.5% 0.01% 0.5%Deionized (52-995-50b) Chitosan (MMW Water Water 0.25% 1xDA-RA) Alginate0.005% Methylene Blue 0.25% Calcium Chloride 0.25% NaTPP 53 0.25% 1 mL0.5% 1% Deionized 1 mL 0.25% 0.01% 1% Deionized (53-989-06) Chitosan(MMW Water + Water + 0.125% 1xDA-RA) 0.1% 0.1% Alginate Sodium Sodium0.005% Benzoate Benzoate Methylene Blue 0.5% Calcium Chloride 0.5% NaTPP0.1% Sodium Benzoate 54 0.375% 1-3 mL 0.75% 0.75% Deionized 1-3 mL 0.25%0.005% 0.75% Deionized (54-1089-37) Chitosan (MMW Water Water 0.125%1xDA-RA) Alginate 0.0025% Methylene Blue 0.375% Calcium Chloride 0.375%NaTPP

As shown in Table 1 above, formulations range in final chitosan contentfrom 0.25% to 0.75%, alginate content from 0.5% to 0.125%, calciumchloride content from 5% to 0.25%, sodium tripolyphosphate content from5% to 0.25%, and methylene blue content from 0.05% to 0.005%. Additionaladditives included dimethylemthylene blue and sodium benzoate.Formulations 12-959-124, 53-989-06, and 54-1089-37 in Table 1 werefurther tested in animal models discussed below.

Example I—In vitro Testing

For the first round of analysis of each tested formulation, samples wereinjected into plastic weigh dishes and allowed to undergo gelation andcrosslinking for one minute prior to analysis. The gel was then analyzedfor both cohesive and adhesive properties by tilting the weigh dish andprodding at the formed gel with tweezers.

Tissue adhesion properties were assessed by spraying the gel over twopieces of fresh bovine liver warmed to 37° C. The gel was allowed tocrosslink for 1 minute before the two gel-coated surfaces were broughtin contact with one another. Gentle pressure was applied to the topsurface as it was slid laterally across the surface of the bottom pieceof tissue. The interaction between the two layers of gel was analyzed toassess the gel's cohesion to itself and adhesion to the tissue to whichit was applied. It was determined that while high concentrations (1.5%)of crosslinkers provided a more robust gel with greater cohesion, itfailed to stimulate increased tissue adhesion and the gel would drag offthe surface of the liver with pressure and collect into a large clump.Thinner formulations with better spray application remained mostly inplace, with at least a thin coating of the applied gel remaining overeach of the tissue surfaces.

The cytocompatibility of the two unreacted components, the mixedunreacted runoff following spray and gelation, and the final gel ofFormulation 12-959-124 (1.5% MMW RA-DA Chitosan+1.5% CaCl2, 0.5%Alginate+0.01% Methylene Blue+1.5% NaTPP, and 0.5% Alginate+0.1° ASodium Benzoate+1.5% NaTPP). Solutions were sterile filtered prior totesting. 500 μL of each solution sample was injected into the center ofa 6 well plate containing L929 mouse fibroblast cells seeded 24 hourspreviously with 100,000 cells/well. To create the gels, 250 μl of eachsolution were injected into a microcentrifuge tube and the resulting gelwas placed in the center of the cell-seeded well using sterile tweezers.Cells were imaged 4 days after the addition of the components, and itwas determined that while the cells in contact with the final gels wereconfluent and growing readily, those in contact with the alginatecomponent alone were surprisingly sparse. Despite this lack ofconfluence in the unreacted alginate samples, there were no signs ofinherent toxicity in these samples.

Degradation of Formulation 12-959-124 was tested by injecting 3 mL ofgel into conical tubes containing PBS (control) or 1.5 μg/mL lysozymesolution. Samples were stored at 37° C. and analyzed weekly over a 28day period for mass loss. As is suggested by the graph of FIG. 1,degradation and clearance from the body is expected within 28 days.

Example 2—In Vivo Testing

Preliminary testing was performed using formulations deemed mostpromising in in vitro experimentation in previously sacrificed pigsprior to proceeding to in vivo experimentation on live animals. Briefly,the surface of the stomach of a pig that had been sacrificed forprevious experimentation was exposed and coated with various testformulations. Gel solutions were allowed to set for 1-2 minutes, afterwhich the surface of the stomach was rubbed against neighboring tissuesto simulate normal peristaltic movements. The gel was assessedqualitatively for its ability to remain in place after contactingsurrounding tissue.

After the first round of porcine testing, Formulation 12-959-124 wasselected for further testing in the in vivo rat model. The formulationwas applied as using a spray applicator assembly designed for theapplication of biomedical materials and commercially available fromNordson Corporation provided with suitable spray tips (i.e., either amanual applicator system using mix tip SA-3674 or an air-assistedapplication system using mix tip SA-3652).

(i) Animal Model Protocol:

In vivo testing was performed by an experienced animal surgeon at theUniversity of Virginia using an established protocol examining adhesionformation between injured abdominal wall and cecum surfaces. Rats wereanesthetized using Ketamine/Xlyazine and matching defects were createdon the abdominal wall and cecum surfaces. The abdominal wall injury wascreated by removing the peritoneum and associated muscle fibers within a1 cm×2 cm area. The cecum defect was created by removing the serosalsheath in a matching 1 cm×2 cm area with 100 wipes using a piece ofgauze. The wounded areas were desiccated by exposure to air for 15minutes, and the animals were then left untreated (control) or treatedwith the hydrogel formulation in accordance with the invention sprayedover each of the injured sites until visual coverage was achieved. Thegel was allowed to crosslink for approximately one minute prior toclosure. Following treatment, the cecum was placed adjacent to theabdominal wall injury site and the incision was closed with sutures andstaples. Rats were bandaged and housed individually until analysis.

Animals were sacrificed and analyzed at Day 7 based on the adhesionscoring system shown in Table 2 below.

TABLE 2 Adhesion Scoring Scale Score Extent Tenacity Type 0    0% Nonenone 1   <25% Easily lysed filmy, no vessels 2 25-50% Lysed w/ Tractionopaque, no vessels 3 50-75% Requires dissection opaque, small vessels 4  >75% opaque, large vessels

Analysis of the sacrificed animal was carried out by carefully openingthe abdominal cavity to prevent the disruption of any adhesions that mayhave formed. The implant site was observed macroscopically for evidenceof gel residence, reactivity of tissue, inflammatory response, andadhesion formation between the cecum, abdominal wall, and surroundingmesentery, viscera, and fat. Though the model is designed to onlyexamine adhesion formation between the abdominal wall and cecum surfaces(Ab-Cec, primary adhesions), the adhesions that occurred between theabdominal wall and other internal tissues (Ab-Int), and cecum to otherinternal tissue (Cec-Int), known as secondary adhesions, were alsoassessed. The adhesions for each pair of tissue were characterizedaccording to the extent of wounded surface covered (score 0-4), thetenacity of the adhesion formed (score 0-3), and the type of adhesionformed (0-4). Therefore, a maximum score of 11 was possible for eachtissue pair combination. The “scaled adhesion score” for each tissuegroup was also examined in which the actual percentage of the injuredsurface covered by the adhesion (0-1) was multiplied by the tenacityscore (0-3) of the adhesion. This analysis resulted in a continuousscoring system between 0-3 for each tissue group and enabled a morecomprehensive understanding of the actual size and strength of theadhesions that formed from various treatment groups.

(ii) Preliminary in vivo testing:

Preliminary animal testing was conducted using Formulation 12-959-124 (a1:1 mixture of 1.5% MMW 1×DA-RA Chitosan/1.5% Calcium Chloride and 0.5%Sodium Alginate/0.01% Methylene Blue/1.5% Sodium Tripolyphosphate)sprayed over the injured tissue surfaces using a manual spray applicatorwith mix tip SA-3674 from Nordson Corporation. Animals received 1-3 mLof each gel sprayed over each injury site. There were no indications ofimmune response or tissue reaction to the implanted product over theseven day test period

Untreated control animals demonstrated consistent formation of theprimary adhesion. The average primary adhesion score for the controlswas 5.75±2.43, and the average total adhesion score was 7.63±3.50. Thosesamples treated with the sprayable hydrogel formulation in accordancewith the invention demonstrated a statistically significant decrease inprimary adhesion formation, with an average primary adhesion score of1.63±2.60. There was, however, a significant increase in secondaryadhesion formation to the injured cecum surface, with a score of3.50±2.19. The total adhesion score for treated animals was 5.44±2.63.All of these scores are presented below in Table 1 below.

TABLE 1 Average adhesion score ± standard deviation for animals in thefull study Ab-Cec Ab-Int Cec-Int Total Control (n = 8) 5.75 ± 2.43 1.38± 1.19 0.5 ± 0.93 7.63 ± 3.50 Spray (n = 16) 1.63 ± 2.60 0.31 ± 0.87 3.5± 2.19 5.44 ± 2.63

The data presented in Table 1 indicate that treatment with the sprayablehydrogel according to the invention decreases the incidence and severityof adhesions that form between the abdominal wall and cecum injurysites. As noted previously, an increase in the incidence of secondaryadhesions was observed between the cecum surface and other internaltissues. This is hypothesized to be due to the fact that a majority ofthe control animals formed the primary adhesion between the abdominalwall and cecum injury sites, leaving these tissues unable to formsecondary adhesions because the injured sites are already occupied. Byeffectively preventing the primary adhesion from forming with theapplication of the hydrogel material, this surface has been freed forthe formation of secondary adhesions. There is no suitable control forsecondary adhesion formation.

(ii) Secondary In Vivo Testing:

Using the air-assisted spray tip SA-3652 applied at 10 psi, only 0.5-1.5mL of the hydrogel could be delivered and still provide sufficientcoverage over both injury sites. This reduction in the total amount ofhydrogel (from 2-6 mL down to 0.5-1.5 mL) required to protect theinjured tissue surfaces successfully resulted in a decrease in primaryadhesion formation and no longer increased the likelihood of secondaryadhesions forming on the cecum surface as was typical in previousapplications during preliminary in vivo testing. Statistical analysis asshown in FIGS. 2A and 2B showed that the decrease in total and scaledadhesion scores for both the primary and total adhesions arestatistically significant.

In an effort to better mimic actual in vivo application, “fullapplication” of the hydrogel has been investigated. In a fullapplication, the hydrogel material is sprayed over both injured surfacesas well as on the surfaces immediately surrounding each injury site fora total volume of 1-2 mL hydrogel applied per animal (using the airassist spray tip). This increased application resulted in improvedprotection of the abdominal wall, eliminating the incidence of secondaryadhesions there and maintaining a statistically significant decrease inthe incidence and strength of primary adhesions. Secondary adhesions tothe cecum surface remain statistically unchanged as compared tocontrols, and total adhesion scores were again reduced significantly(See FIGS. 3A and 3B).

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not to be limited to thedisclosed embodiment, but on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope thereof.

What is claimed is:
 1. A biologically acceptable surgical barriermaterial comprised of a polyelectrolytic complex of chitosan and sodiumalginate.
 2. The material of claim 1, wherein the chitosan isdeacetylated in an amount between about 40 to about 60%.
 3. The materialof claim 2, wherein the chitosan has a molecular weight (Mw) between50,000 and 375,000 g/mol.
 4. The material of claim 1, further comprisingan ionic crosslinker.
 5. The material of claim 4, wherein the ioniccrosslinker is selected from the group consisting of calcium chlorideand sodium tripolyphosphate.
 6. The material as in claim 1, whichfurther comprises a colorant.
 7. A sprayable biologically acceptablesurgical barrier material system comprised of a first componentcomprising chitosan and a second component comprising sodium alginate,wherein mixing of the first and second components forms apolyelectrolytic complex of the chitosan and sodium alginate.
 8. Thematerial system of claim 7, wherein the chitosan is deacetylated in anamount between about 40 to about 60%.
 9. The material system of claim 8,wherein the chitosan has a molecular weight (Mw) between 50,000 and375,000 g/mol.
 10. The material system of claim 7, wherein at least oneof the first and second components further comprise an ioniccrosslinker.
 11. The material system of claim 10, wherein the firstcomponent comprises calcium chloride as an ionic crosslinker.
 12. Thematerial system of claim 10, wherein the second component comprisessodium tripolyphosphate as an ionic crosslinker.
 13. The material systemas in claim 7, which further comprises a colorant.
 14. The materialsystem as in claim 7, wherein the second component is comprised of asolution comprised of 0.25 to about 0.5% w/v of the sodium alginatehaving a molecular weight of from about 75,000 to about 500,000 g/mol.15. The material system of claim 14, wherein the second componentcomprises between about 0.5 to about 1.5 wt. %, based on total weight ofthe second component, of sodium tripolyphosphate.
 16. A method offorming a biologically acceptable barrier material at a surgical sitecomprising: (i) mixing the first and second components of the barriermaterial system according to claim 7 to form a sprayable mixturethereof, (ii) directing the sprayable mixture toward the surgical site,and (iii) allowing a surgical barrier material formed of apolyelectrolytic complex of the chitosan and sodium alginate to form insitu at the surgical site.
 17. The method according to claim 16, whereinsteps (i) and (ii) are practiced using a manual or a gas-assisted mixingand spray applicator.
 18. The method according to claim 16, whereinsteps (i) and (ii) are practiced with a gas-assisted mixing and sprayapplicator using atmospheric air or carbon dioxide gas at an atomizingpressure of between about 5 psi to about 15 psi.
 19. The methodaccording to claim 16, wherein at least one of the first and secondcomponents comprises a colorant.
 20. The method according to claim 16,wherein at least one of the first and second components further comprisean ionic crosslinker.
 21. The method according to claim 20, wherein thefirst component comprises calcium chloride as an ionic crosslinker. 22.The method according to claim 20, wherein the second component comprisessodium tripolyphosphate as an ionic crosslinker.